Microvesicle-Based Compositions and Methods

ABSTRACT

Methods and compositions for diagnosis and/or analysis of a condition in a mammal are disclosed in which RNA from microvesicles is enriched and differentiated to so obtain a result that is indicative of the condition of tissue or organ from which the microvesicle originated. In especially preferred embodiments, the condition is a neoplastic disease of a human and can be identified and staged by differential analysis of one or more distinct RNAs, optionally together with identification and analysis of a non-RNA component of the microvesicle.

This application is a continuation-in-part application of our USapplication with the Ser. No. 11/569,757, filed Nov. 29, 2006, which isa national phase application of our International application with theserial number PCT/US05/10674, filed Mar. 30, 2005, which claims priorityto our US provisional application with Ser. No. 60/576,395, filed Jun.2, 2004. This application also claims priority to our US provisionalapplication with Ser. No. 61/074,218, filed Jun. 20, 2008. All prioritydocuments are incorporated by reference herein.

FIELD OF THE INVENTION

The field of the invention is detection and/or analysis of RNA inmicrovesicles and their use in diagnosis, prognosis, and/or research ofdiseases and other conditions.

BACKGROUND OF THE INVENTION

Microvesicles were historically regarded as cellular debris with noapparent function. However, a growing body of experimental data hassuggested that microvesicles have numerous biological activities. Forexample, platelet-derived microvesicles were shown to stimulate selectedcells via surface proteins on the microvesicles (e.g., Thromb. Haemost.(1999), 82:794, or J. Biol. Chem. (1999), 274:7545). In other examples,specific effects of bioactive lipids in platelet microvesicles oncertain target cells were reported (e.g., J. Biol. Chem. (2001), 276:19672; or Cardiovasc. Res. (2001), 49(5):88). In still further examples,platelet microvesicles increased adhesion of mobilized CD34+ endothelialcells by transfer of certain microvesicle surface components to themobilized cells (e.g., Blood (2001), 89:3143).

More recently, microvesicles have also been shown to comprise RNA thatat least in part appeared to reflect the RNA content of the cell fromwhich they originate. Microvesicles have also been shown to havesignificant biological effect on other cells, probably due to the RNApresent in the microvesicles, and various examples and aspects for suchmicrovesicles are described in our commonly owned Internationalapplication (WO2005/121369), which expressly forms part of thisapplication.

In further known reports, microvesicles were described as includingnon-coding miRNA (microRNA) that could potentially interfere or regulategene expression in cells with which such microvesicles merge (PLoSNovember 2008, Vol. 3(11), e3694). Other reports discuss in vitrocell-to-cell signaling via exosomal RNA (Cell Adh Migr 1:3, 156-158;2007; Cancer Immunol Immunother 2006 July; 55(7):808-18; Blood. 2007Oct. 1; 110(7):2440-8.). It was also shown that while some exosomal RNAwas functional and translatable in a recipient cell, many of the RNAmolecules present in the exosomes were not present in the cytoplasm ofcells from which the exosomes were though to have originated (Nat CellBiol. 2007 June; 9(6):654-9). U.S. Pat. No. 6,916,634 teaches that whileRNA is generally instable in serum and readily hydrolyzed by RNAses,other RNA is resistant to RNAse attack, presumably due to its varyingassociation with circulating particles. Remarkably, the '634 patentelaborates on the chemically and structurally highly diverse nature ofthe RNA associated particles (presumably due to their diverse origin andmanner of generation) and thus concludes that serum RNA is best isolatedin an indiscriminate manner. Thus, even though membrane associated orvesiculated RNA has more recently been reported, there is a large bodyof contradictory data and hypotheses with respect to the nature,quality, availability, and origin/manner of generation of microvesicles.

Consequently, the enormous diagnostic potential of RNA-containingmicrovesicles has not been fully recognized in the art, and there isstill a need for microvesicle-based diagnostic compositions and methodsin which RNA from microvesicles is enriched and differentiated to soobtain a result that is indicative of the condition of tissue or organfrom which the microvesicle originated.

SUMMARY OF THE INVENTION

According to the present inventive subject matter, microvesicles areemployed in various diagnostic, prognostic, and/or analytic compositionsand methods in which specific RNA content of microvesicles andoptionally at least one more additional information bearing component ofthe microvesicle are used to obtain cell-, tissue-, organ-, and/ordisease-specific information.

In one especially preferred aspect of the inventive subject matter,method of analyzing a biological sample (e.g., plasma or serum) of amammal in which microvesicles are obtained from a living donor mammal(preferably human) that include a plurality of distinct RNA molecules.The microvesicles are then enriched and differentiated to produce aprimary result based on one or more distinct RNA molecules, a secondaryresult based on at least two distinct RNA molecules, and/or a ternaryresult based on sub-segregated proxysomes and one or more distinct RNAmolecules. Of course, it should be appreciated that the RNA analysis inthe differentiation may include analysis of a single RNA, of at leasttwo distinct RNAs, and in some aspects even an entire RNA profile(typically obtained by analysis of an array of multiple distinct DNA orRNA). The so obtained results are then correlated with a diagnosis(e.g., cancer or a clinical stage of a cancer, including pre-cancerousstages) or prognosis/diagnosis of a condition of the mammal.

Most typically, the distinct RNA molecule(s) in the primary resultis/are RNA that is overexpressed, underexpressed, and/or mutated,wherein the change in expression and/or the mutation is characteristicfor the condition. Similarly, one of the two distinct RNA molecules inthe secondary result is an RNA that are overexpressed, underexpressed,and/or mutated, wherein the change in expression and/or the mutation ischaracteristic for the condition, while the other of the distinct RNAmolecules in the secondary result is an RNA that is uniquely expressedin a specific tissue or organ of the mammal. The sub-segregatedproxysome population is preferably obtained by isolating the proxysomepopulation based on a surface molecule specific for origin of theproxysome population, and the distinct RNA molecule in the ternaryresult is RNA that is overexpressed, underexpressed, and/or mutated,wherein the change in expression and/or the mutation is characteristicfor the condition.

With respect to the RNA that is overexpressed, underexpressed, and/ormutated, it is typically preferred that the RNA encodes MMP11, BCAR1,ERBB2, MKI67, PLAU, and/or TP53 where the condition is breast cancer; orencodes FGFR1, KRAS2, TGFBR2, MAP2K4, and/or CDKN2A where the conditionis pancreatic cancer; or encodes KLK3, ERBB2, FGF8, PSCA, and/or CAV1where the condition is prostate cancer; or encodes BAX, SLC2A1, PTGS2,MUC1, and/or RUNX3 where the condition is gastric cancer; or encodesBCL10, PAP, SPARC, CD44, and/or TP53 where the condition is livercancer; or encodes a stem cell marker, and especially an adult stem cellmarker (e.g., CD33, CD44, CXCR4, CXCR4+, lin-, CD45−, Oct-4, Nanog,SCA1, 7-AAD), where the condition is a cancer.

With respect to the RNA that is uniquely expressed in the specifictissue or organ it is preferred that the RNA encodes DCD, SCGB2A2,and/or ANKRD30A where the specific tissue or organ is breast tissue; orencodes UCN3, IPF1, and/or REG1B where the specific tissue or organ is apancreas; or encodes UPK3A, SEMG1, and/or PRAC where the specific tissueor organ is prostate tissue; or encodes GAST, GKN1, and/or TFF2 wherethe specific tissue or organ is gastric tissue; or encodes GYS2, F9,and/or HRG where the specific tissue or organ is a liver.

It is further generally preferred that the microvesicles are enrichedvia aggregation by interlinking the microvesicles with an interlinkingcomposition (e.g., annexin V, fibrin, or an antibody or antibodyfragment against a tetraspanin, ICAM-11, or CD86). Consequently,microvesicles especially contemplated herein have a membrane compositionsuch that phosphatidylserine is on the outside of the membrane. It isfurther generally preferred that the primary or secondary result areobtained using a quantitative rtPCR for the distinct RNA molecule or viamicroarray technology.

Thus, viewed from a different perspective, a method of staging amammalian neoplasm (which also includes staging of precancerous lesionsor growth) will include a step of obtaining a whole blood fraction(e.g., serum or plasma) that includes a plurality of microvesiclescomprising a plurality of distinct RNA molecules. In another step, themicrovesicles are enriched (e.g., via centrifugation and/or aggregation)and differentiated (e.g., using quantitative rtPCR) to produce a primaryresult based on at least one distinct RNA molecule, a secondary resultbased on at least two distinct RNA molecules, and/or a ternary resultbased on a sub-segregated proxysome population and at least one distinctRNA molecule. The results are then correlated with a stage of theneoplasm in the mammal, typically via comparison with a referenceresult.

Among other neoplasms, especially contemplated neoplasms include acutelymphoblastic leukemia, bladder cancer, breast carcinoma, cervicalcancer, colorectal cancer, lung cancer, ovarian cancer, and pancreaticadenocarcinoma. Therefore, one of the at least two distinct RNAmolecules in the secondary result or one of the distinct RNA moleculesin the primary or ternary result will be an RNA that encodes ERBB2.

Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph depicting the results for in vitro expression ofHer2-RNA in selected cell lines.

FIG. 2A is a photograph of an agarose gel with Her2-RNA amplificationproducts of RNA from microvesicles that were isolated from culturesupernatants of the selected cell lines.

FIG. 2B is a graph depicting the quantitative difference of theamplification products of FIG. 2A.

FIG. 3A is a photograph of an agarose gel with Her2-RNA amplificationproducts of RNA from microvesicles that were isolated from murine serumof mice that developed tumors derived from one of the selected celllines.

FIG. 2B is a graph depicting the quantitative difference of theamplification products of FIG. 3A.

FIG. 4A is a photograph of an agarose gel with Her2-RNA amplificationproducts of RNA from microvesicles that were isolated from human sera ofpatients with confirmed breast cancer diagnosis.

FIG. 4B is a graph depicting the quantitative difference of theamplification products of FIG. 3A.

DETAILED DESCRIPTION

The inventor has discovered that mammalian, and especially humanmicrovesicles can be employed as a proxy diagnostic tool in the analysisand/or diagnosis of a cell, tissue, organ, or even multi-organ systemwhere RNA and/or multiple non-RNA components (e.g., soluble and/ormembrane proteins, lipids, etc.) from the microvesicles are correlatedwith status and/or health of the cell, tissue, organ, or evenmulti-organ system from which the microvesicle originated. Based on theinventor's findings that the RNA content of the microvesicles isentirely or almost entirely representative of the cellular RNA of thecell from which the microvesicle originated, numerous uses are nowenvisioned. As microvesicles are shed by all cells in relatively highnumbers, and as microvesicles are also present in equally high number inblood, the inventor concluded that, inter alia, gene expression,soluble/membrane proteins, and membrane composition of various cells,tissues, and organs can be easily determined by analyzing thecorresponding microvesicles, where those microvesicles are obtained froma biological fluid, and especially blood.

The term “microvesicle” as used herein refers to a membranaceus particlehaving a diameter (or largest dimension where the particle is notspheroid) of between about 10 nm to about 5000 nm, more typicallybetween 30 nm and 1000 nm, and most typically between about 50 nm and750 nm, wherein at least part of the membrane of the microvesicle isdirectly obtained from a cell. Therefore, especially contemplatedmicrovesicles include those that are shed from a donor cell, and willtypically also include exosomes. Therefore, and depending on the mannerof generation (e.g., membrane inversion, exocytosis, shedding, orbudding), the microvesicles contemplated herein may exhibit differentsurface/lipid characteristics. Viewed from a different perspective, themicrovesicles suitable for use in the present inventive subject mattermay originate from cells membrane inversion, exocytosis, shedding,blebbing, or budding. Most typically, microvesicles suitable for useherein will be identifiable by having phosphatidylserine, a tetraspanin,ICAM-1, and/or CD86 on the outer surface. In contrast, a liposome madefrom isolated lipids will not include lipids or other componentsobtained from a cell-membrane, and is not considered a microvesicleunder the definition used herein. Therefore, most typically,contemplated microvesicles will have a lipid bilayer structure and arenot multi-lamellar.

Moreover, it is preferred that the microvesicles are generated from adifferentiated cell, and most preferably from a terminallydifferentiated cell (i.e., a cell that has reached the end of itsdifferentiation pathway). Therefore, microvesicles generated by blastcells, progenitor cells, and stem cells are excluded in at least some ofthe embodiments herein. However, and especially where the microvesiclesare generated from a cancer cell, and where that cancer cell is derivedfrom an adult stem cell (as opposed to an embryonic stem cell) lessdifferentiated cells are also deemed suitable sources for themicrovesicles. It is especially preferred that the microvesicles arederived from cells that are diseased (e.g., neoplastic cell, infectedcell, cell in an infected organ) or subject to an abnormal condition(e.g., metabolic abnormality, cell exposed to a drug, and more typicallya drug that preferentially affects the cell relative to other cells, ordietary toxin, or damaged by free radicals). In still furthercontemplated aspects, the microvesicles are generated from a healthy,but ageing cell, i.e., a cell that has reached at least 50%, and moretypically at least 70% of its ordinary number of cell divisions(Hayflick limit). It should be noted that synaptosomes (vesicularentities in the synaptic gap, but not present in the generalcirculation) and microvesicles that are formed from platelets areexpressly excluded.

Thus, the RNA content of the microvesicles is thought to becharacteristic of a particular condition (e.g., disease, age, stress,response to a chemical compound, senescence, inflammation, infection[e.g., viral, microbial, parasitic], immune status, regeneration,rejection, etc.). Non-RNA information bearing components inmicrovesicles contemplated herein include various proteins (e.g.,receptor, ligand, glycoprotein, etc.) that are associated with themicrovesicle, which may be at least partially embedded in the membrane(or even be entirely enclosed), specific lipids or glycolipids that areassociated with the microvesicle, and/or a further nucleic acid (DNAand/or RNA) associated with the microvesicle. Thus, and especially wherethe non-RNA information bearing component is specific to a particularorgan, tissue, and/or cell, analysis of the condition of the particularorgan, tissue, and/or cell can be obtained in a highly simplified andeven multiplexed manner. Consequently, and among other advantages, itshould be especially appreciated that a serum or blood based test can beperformed that provides (even multiple) organ specific results withoutthe need for an otherwise required organ-specific biopsy. Still further,it should be noted that the methods contemplated herein will allowvirtually unlimited and repeated real time access to an expressionprofile of the same tissue or organ.

Consequently, methods according to the inventive subject mater willtypically include the following sequence (in which one or more steps maybe combined):

Isolation→Differentiation→Analysis.

More specifically, and in one particularly preferred example, theinventor contemplates a method of analyzing a biological sample of amammal in which a plurality of microvesicles that include distinct RNAmolecules is first obtained from a living donor mammal. Themicrovesicles are then enriched and differentiated to produce a primaryresult based on one or more distinct RNA molecules (which are mosttypically a RNA that are uniquely expressed and/or mutated as a functionof the condition of the cell from which it derived), a secondary resultbased on at least two distinct RNA molecules (with one of the moleculesbeing RNA that is uniquely expressed in a specific tissue or organ, andwith the other molecule being RNA that is uniquely expressed and/ormutated as a function of the condition of the cell from which itderived) and/or a ternary result based on a sub-segregated proxysomepopulation (e.g., based on a specific surface marker of the cell fromwhich the microvesicle was derived) and one or more distinct RNAmolecules (most typically a RNA that is uniquely expressed and/ormutated as a function of the condition of the cell from which itderived). The primary, secondary, and/or ternary results are thencorrelated with a diagnosis or prognosis of a condition of the mammal.

Most typically, the microvesicles are obtained from whole blood, serum,plasma, or any other biological fluid, including urine, milk, tears,spinal fluid, amniotic fluid, etc., which are preferably obtained from aliving mammal, and most preferably within the time limit acceptable forprocessing biological fluids for later clinical analysis. Alternatively,in less preferred aspects, microvesicles may also be obtained fromstored materials (e.g., biological fluids, tissues, organs, etc.),wherein the time between obtaining the biological fluid, tissue, ororgan, and enrichment of the microvesicles from the sample may be atleast 12 hours, at least 24 hours, at least 2-5 days, or even at leastone or more weeks. Such storage may even include storage at reducedtemperature (e.g., 4° C.) or even storage in frozen form. Similarly,microvesicles may also be obtained from an in vitro source, and mosttypically from cell or tissue culture, or even organ culture. In yetfurther contemplated aspects, it should be noted that deceased donorsare also deemed suitable as a source for the microvesicles. Mostcommonly, however, microvesicles will be obtained from blood, which maybe immediately or within a few hours (less than 12 hours) processed toform serum or plasma, which is then either stored, shipped, and/orfurther processed o enrich the microvesicles.

With respect to isolation or enrichment of microvesicles it iscontemplated that all known manners of isolation of microvesicles aredeemed suitable for use herein. As used herein, the terms “isolation” or“isolating” in conjunction with microvesicles are interchangeably usedwith the terms “enrichment” or “enriching”, and refer to one or moreprocess steps that result in an increase of the fraction ofmicrovesicles in a sample as compared to the fraction of microvesiclesin the obtained biological sample. Thus, microvesicles may be purifiedto homogeneity, purified to at least 90% (with respect tonon-microvesicle particulate matter), less preferably at least 80%, evenless preferably at least 50%, and least preferably at least 20% (or evenless). For example, physical properties of microvesicles/proxysomes maybe employed to separate them from a medium or other source material, andespecially preferred physical methods include separation on the basis ofelectrical charge (e.g., electrophoretic separation), size (e.g.,filtration, molecular sieving, etc), density (e.g., regular or gradientcentrifugation), Svedberg constant (e.g., sedimentation with or withoutexternal force, etc). Alternatively, or additionally, isolation may bebased on one or more biological properties, and especially suitableisolation methods may employ surface markers (e.g., for precipitation,reversible binding to solid phase, FACS separation, specific ligandbinding, non-specific ligand binding such as annexin V, etc.). In yetfurther contemplated methods, the microvesicles may also be fused usingchemical and/or physical methods, including PEG-induced fusion and/orultrasonic fusion.

Viewed from a different perspective, enriching can be done in a generaland non-selective manner (typically including serial centrifugation),and may be performed by aggregation where the microvesicles areinterlinked with an interlinking composition (e.g., annexin V, fibrin,or an antibody or fragment thereof against at least one of atetraspanin, ICAM-1, and CD86). Alternatively, enriching can be done ina more specific and selective manner (e.g., using tissue or cellspecific surface markers). For example, specific surface markers may beused in immunoprecipitation, FACS sorting, bead-bound ligands formagnetic separation etc. Such isolation or enrichment willadvantageously allow obtaining cell-, tissue-, organ-, and/or diseasespecific information without the need for direct access to the cell,tissue, or organ under investigation. The specific RNA that is enclosedin a general population of microvesicles is therefore generally termed“vesiculated RNA”, while RNA of a specific type of microvesicle (e.g.,microvesicle generated by hepatocyte) is termed “proxy RNA”, andmicrovesicles that are specific to a single type of origin are referredto as “proxysomes”. Such proxysomes will consequently have an RNAcontent and membrane composition (especially in terms of surfacemarkers, but also to at least some degree in terms of lipid composition)that is consistent with the RNA content of a cell from which theproxysome is produced.

Therefore, and especially where the microvesicles/proxysomes areisolated from a biological source (e.g., whole blood or serum) or mixedcell or tissue culture, it should be noted that the isolation mayproduce a heterogeneous population of microvesicles/proxysomes withrespect to the cell-/tissue-, and/or organ-type from which themicrovesicles/proxysomes were produced. On the other hand, andespecially where a specific surface marker was used in the isolation ofthe microvesicles/proxysomes, the isolated population may already behomogenous with respect to the cell-/tissue-, and/or organ-type fromwhich the microvesicles/proxysomes were produced. In still furthercontemplated aspects, the mechanism of release of the vesicles from thecell may be used to further differentiate, even among proxysomes.However, it should be noted that all vesicular architectures (e.g.,inside-out, etc.) are deemed suitable for use herein.

As a population of microvesicles obtained from a biological fluid willtypically represent a plurality of cells, tissues, and organs,differentiation of the heterogeneous population is often desirable toobtain a cell, tissue, or organ specific result. Differentiation of themicrovesicles is typically dependent on the type of cell, tissue, ororgan under investigation, and conceptually at least three distinctapproaches can be taken.

First, where a condition (e.g., cancer, infection, senescence,inflammation, etc.) of a cell, tissue, or organ is associated with aunique and distinguishable expression profile (e.g., over- orunderexpression) of a gene and/or with a specific mutation, a meaningfulresult may be based on the expression profile and/or specific sequenceof one or more distinct RNA molecules without further normalization ofthe signal. For example, where the condition is breast cancer, severalgenes are often misregulated, and presence of high quantities of ERBB2will be indicative of breast cancer. Similarly, certain types ofleukemia (CML) are associated with a specific fusion mutant (Bcr/Abl)that is specifically associated with the leukemia. Thus, a primaryresult can be obtained based on the expression profile and/or specificsequence of one or more distinct RNA molecules without physicalsub-segregation of the microvesicles.

Second, a condition (e.g., cancer, infection, senescence, inflammation,etc.) of a cell, tissue, or organ may be associated with a unique anddistinguishable cell, tissue, or organ type and may therefore bespecifically characterized by normalizing an expression profile and/orspecific sequence of one or more distinct RNA molecules against theexpression profile and/or specific sequence of one or more furtherdistinct RNA molecules that are uniquely present or expressed in thespecific cell, tissue, or organ. Thus, a secondary result can beobtained based on the expression profile and/or specific sequence of atleast two distinct RNA molecules without physical sub-segregation of themicrovesicles.

Third, a condition (e.g., cancer, infection, senescence, inflammation,etc.) of a cell, tissue, or organ may be associated with a unique anddistinguishable cell, tissue, or organ type and may therefore bespecifically characterized by physical sub-segregation (infra) of themicrovesicles prior to analysis of the expression profile and/orspecific sequence of one or more distinct RNA molecules in thesub-segregated microvesicles.

Therefore, in some aspects of the inventive subject matter,differentiation is preferably done using one or more additionalinformation bearing components in and/or on the microvesicle tocorrelate the result with the additional information to so obtainnormalized cell-, tissue-, organ-, and/or disease-specific information.Viewed from a different perspective, differentiation may thereforeinclude a sub-segregation of a subset of proxysomes from themicrovesicles, and/or a determination of a reference marker that isspecific to a subset of proxysomes of the isolated population. It shouldbe especially appreciated that the differentiation may also includedetection or use of a component (or portion thereof) on a microvesicleor proxysome that is otherwise not accessible on the cell. Such isespecially true where the microvesicle or proxysome has an inside-outarchitecture that exposes on the outside of the microvesicle orproxysome the component that has an orientation normally orientedtowards the interior of the cell from which the vesicle is produced.

For example, where differentiation is based on molecular analysis ofgene expression of a gene that is significantly associated with aparticular disease, differentiation may be based on the unique sequence,expression profile, mutation, or other character of a particular RNA andthus will typically not require physical subsegregation and/ornormalization against other RNA and/or protein markers. Thus, in atleast some instances, presence, abundance, and/or sequence of one ormore RNA molecules in the microvesicle population can be directlyattributed to a specific disease or condition of a cell, tissue, ororgan. For example, where a patient is suspected of breast cancer thepresence, specific sequence, and/or expression rate of a relevantoncogene (e.g., BRCA, Her2/neu, etc.) in a microvesicle will be usefulas a proxy marker for the cancer as such particular RNA is normally notpresent in measurable quantities in microvesicles.

Alternatively, and especially where an RNA under investigation ispresent in diseased (or otherwise compromised) as well as healthy cells,tissues, or organ, differentiation may be based on normalization of theRNA under investigation against one or more other constituent parts ofthe microvesicles/proxysomes. Therefore, it should be appreciated thatdifferentiation can be performed on a non-segregated pool ofmicrovesicles, and even on the source material where the microvesiclesare present. In such case, it is generally preferred that the additionalinformation bearing component is specific to a particular subset ofproxysomes. For example, it is of interest to analyze microvesicles frommammary gland tissue, a gene may be identified that is uniquely (greateror equal 90%) or predominantly (greater or equal 75%) expressed in themammary gland tissue. As the proxysome RNA content is an at leastpartial, and in many cases a complete representation of the RNA contentof the mammary gland cell from which the proxysome is derived, themammary gland proxysome is expected also to comprise the uniquely orpredominantly expressed gene. For example, it is known that dermcidinis >95% selectively expressed in mammary gland tissue and therefore alsoexpected to be uniquely present in mammary gland proxysomes. There arenumerous genes known in the art that are uniquely or predominantlyexpressed in specific cells and organs, and suitable genes with suchexpression can be found, inter alia, at the TIGER database(http://bioinfo.wilmer.jhu.edu/tiger/; incorporated by referenceherein).

Alternatively, or additionally, numerous known non-nucleic acidcomponents can be used to differentiate and may include disease specificmarkers (e.g., tumor specific markers such as CEA), organ specificmarkers (e.g., PSA), and/or cell-specific markers (e.g., CD4 forT-helper cells), etc. These additional information bearing componentsmay then be used for physical subsegregation in a manner as describedabove. However, it is also noted that so long as these additionalinformation bearing components are specific to the desired subpopulationof proxysomes, no physical subsegregation may be required, and the RNAinformation (infra) may be normalized or otherwise parameterized usingthe additional information bearing component. Thus, it should berecognized that molecular analysis for differentiation can be done withhigh specificity to a particular cell type, tissue type, and/or organtype.

Physical subsegregation of a subset of proxysomes may be performed usingantibodies or other binding agents that binds selectively to a componentthat is specific to particular cell, tissue, or organ. Such componentsare typically cell-, tissue-, or organ-specific marker, receptors,structural components, glycoproteins, CDs, etc., all of which may befurther derivatized. Suitable organ, tissue, and cell specific surfacemarkers are well known in the art and can be found in numerouspublications (e.g., for natural receptors see e.g., Cell SurfaceReceptors: A Short Course on Theory and Methods by Lee E. LimbirdSpringer; 2nd ed. edition (Dec. 31, 1995), ISBN-10: 0792338391; forsynthetic peptides to selected cells, see e.g., Curr Opin Chem Biol.2000 February; 4(1):16-21). Thus, it should be appreciated thatsubsegregation can be performed specific to a cell type, tissue type,and/or organ type.

Most typically, physical subsegregation will involve use of a solidphase to which a compound is bound that (preferably releasably) binds toone or more of the component that is specific to particular cell,tissue, or organ. For example, where the solid phase is a multi-wellplate, proxysomes may be separated into separate wells. Where the solidphase is a magnetic or color coded bead, the proxysomes may be isolatedusing a magnet or light activated sorter. Similarly, the solid phase maybe a dipstick or membrane and separation will (preferably releasably)bind the proxysomes on the solid phase. In still further contemplatedaspects, subsegregation may also be performed on an array in whichantibodies are coupled to the surface of the array in predeterminedpositions to so specifically bind distinct proxysomes in distinct andpredetermined positions on the array. Detection of the bound proxysomesmay then be performed in a non-specific manner (e.g., using a labeledform of annexin V) or a specific manner (e.g., using a labeled antibodyagainst another surface marker of the proxysome).

Of course, it should be recognized that subsegregation may be done afterisolation of a population of microvesicles, but may also be directlyperformed on the source material of the microvesicles/proxysomes. Insuch case, the steps of isolation and subsegregation are combined. Mosttypically, and depending on the surface marker used, the subsegregationwill produce a subpopulation of proxysomes that is substantiallyhomogeneous (i.e., at least 70%, more typically at least 80%, mosttypically at least 90%) with respect to the tissue from which theyoriginate (e.g., microvesicle from liver, prostate, mammary gland,etc.). It should further be

The specific RNA of the proxysome of interest can most typically beanalyzed using all known manners of RNA analysis, and particularlypreferred manners include rtPCR, quantitative PCR (rt or otherwise),primer extension analysis (with or without prior amplification), and allsolid-phase based methods (e.g., using arrays, magnetic or color codedbeads, etc.). While not limiting to the inventive subject matter, it isgenerally preferred that the specific RNA is (1) native to the cell,tissue or organ, i.e., not introduced (directly or indirectly) by apathogen, and/or (2) specific or indicative to a predisposition,condition, disease, response to a stimulus, and/or pathogen. Forexample, suitable specific RNAs include genes that are specifically andexclusively expressed or overexpressed in cells, tissues, or organsaffected with a neoplastic disease, a metabolic disease, inflammation,senescence, hypoxia, an infection, and/or inheritable disease.

For example, especially suitable specific RNAs are those known to havean association with a particular disease and especially with cancer.There are numerous such RNAs and genes known in the art, and all ofthose are deemed suitable for use herein. Among other sequences, RNAspecific for breast cancer includes RNA that encodes MMP11, BCAR1,ERBB2, MKI67, PLAU, and/or TP53, RNA specific for pancreatic cancerincludes RNA that encodes FGFR1, KRAS2, TGFBR2, MAP2K4, and/or CDKN2A,RNA specific for prostate cancer includes RNA that encodes KLK3, ERBB2,FGF8, PSCA, and/or CAV1, or RNA specific for gastric cancer includes RNAthat encodes BAX, SLC2A1, PTGS2, MUC1, and/or RUNX3, RNA specific forliver cancer includes RNA that encodes BCL10, PAP, SPARC, CD44, and/orTP53, and RNA found for various cancers includes RNA that encodes adultstem cell markers (e.g., CD33, CD44, CXCR4, CXCR4+, lin-, CD45−, Oct-4,Nanog, SCA1, and/or 7-AAD. Further numerous cancer related genes/RNA canbe found in various publicly or commercially available database (e.g.,http://www.cancerindex.org/geneweb/clink30.htm, incorporated byreference herein). In still further especially preferred aspects, it iscontemplated that more than one specific RNA is measured to so improve adiagnostic finding. For example, where paired or grouped markers areanalyzed, ERBB2 and VEGFR may be measured in a population of proxysomesfrom mammary gland tissue.

Of course, it should be noted that the RNA of interest need not belimited to an RNA that is associated with a particular disease, but thatsuitable information may be generated from analysis of the expressionpattern on of or more distinct RNAs. For example, it is contemplatedthat total RNA from a non-segregated microvesicle population is analyzedon a chip of other multiplexed platform to so arrive at a genome wideexpression profile. Such expression profile may then be used as a basisfor diagnosis of a condition or disease where an expression profile of ahealthy individual is known. Consequently, it should be appreciated thatfor the first time, one or more systemic expression profiles may beobtained from the same individual (or group of individuals) in a mannerthat does not require multiple biopsies. Similarly, and especially wherethe RNA analysis is normalized against a specific tissue or performed ona sub-segregated population of microvesicles, tissue specific expressionprofiles may be obtained from the same individual (or group ofindividuals) in a manner that does not require biopsies.

While not expressly excluded, it is generally preferred that the RNA isan RNA that is coding and can give rise to a protein via ribosomaltranslation. Therefore, most typically RNA will include a 3′-polyA tailand may further include a 5′-cap structure (typically 7-methylatedguanine nucleotide bound to mRNA via 5′-5′ triphosphate group, but lesscommon structures also deemed suitable and include methylation of the 2′hydroxy-groups of the first 3 ribose sugars of the 5′ end of the mRNA).Furthermore, it should also be noted that while mRNA is deemedespecially useful in conjunction with the teachings presented herein,other forms of RNA are also expressly contemplated herein. For example,suitable RNA for analysis include single stranded and double strandedRNA, heterogenous RNA (hnRNA), small interfering RNA (siRNA), transferRNA (tRNA), ribosomal RNA (rRNA), mitochondrial RNA (mtRNA), capped RNAand uncapped RNA, polyadenylated and non-polyadenylated RNA, etc.

Analysis of the specific RNA and the additional information bearingcomponent is based on the premise that each proxysome will have at leastone RNA specific for a disease/condition and at least one additionalrepresentative component of the cell from which that proxysome wasproduced (e.g., RNA of a tissue-specific expressed gene, a cytoplasmiccomponent, a membrane composition, membrane associated component(typically comprising a protein). Together, the specific RNA and theadditional component (which may be another RNA that is cell- or tissuespecific, a protein, a lipid, or any combination thereof) are thenemployed to analyze a condition (e.g., disease or predisposition, age,reaction to physical or chemical stimulus [e.g., drug, food, radiation,etc.]) in a highly cell-, tissue-, and/or organ-specific manner withoutthe need to directly sample the cell-, tissue-, and/or organ. Analysismay be typically be specific with respect to a set of markers. However,analysis may also be analyzed as a function of time to see fluctuationsin total microvesicles and/or proxysomes or proxysome populations.Analysis may be focused on time course, quantity, and/or associationwith further markers. Thus, analytic results will typically includevariants, quantities, and/or or time course expression of the specificRNA, which will most typically be correlated with one or more resultsobtained from the differentiation (which may be a quantitative measureof mRNA, a peptide, a membrane component, etc.).

With respect to analysis of the additional information bearing componentit should be appreciated that those components can be analyzed (or used)in all known manners. For example, where the additional informationbearing component comprises a peptide, especially preferred mannersinclude use of antibodies (or fragments thereof), ligands (synthetic ornatural), etc., all of which may be labeled or otherwise modified forqualitative or quantitative analysis. Where the additional informationbearing component is a polysaccharide, lectins may be used, and wherethe additional information bearing component is a lipid, specificligands (e.g., annexin V) may be used for quantification. Moreover, andespecially where the additional information bearing component is acompound other than a nucleic acid, it is contemplated that theadditional information bearing component may be used to sub-segregate apopulation of microvesicle into a population that is enriched in onespecific type of microvesicle (enrichment preferably>80%).

Depending on the particular RNA and manner of differentiation, it shouldbe appreciated that the manner of correlation of the results to aspecific diagnosis or condition may vary to at least some degree. Forexample, where the analysis of RNA relies on quantification and/orsequence of a disease-specific gene, correlation may advantageously bedone by comparison of the result(s) with result(s) obtained from amicrovesicle population obtained from a reference patient (which may behealthy, or representative to a specific disease stage or state).Similar correlation can be performed for tests in which the RNA isnormalized against another RNA or non-RNA compound from a microvesicle,or is normalized meg of total RNA/mL of serum, total RNA per mg of MVsproteins isolated from 1 mL of serum, or other preferably fixedparameter.

In like manner, where the RNA is obtained from a population ofproxysomes, correlation is typically performed against a known standardthat is indicative for a particular disease or condition. Of course, itshould be appreciated that the comparison of the results may beperformed one a single RNA basis, on the basis of two or more RNAresults, or in at least some cases, on a genome or organ-specific basis.In such case, the correlation will include comparison of multipleRNA-specific results with multiple reference results, and all possiblecross-relations thereof.

Thus, it should be appreciated that the inventor especially contemplatesa method of confirming and/or staging a mammalian neoplasm in which inone step a whole blood fraction is obtained that includes a plurality ofmicrovesicles comprising a plurality of distinct RNA molecules. Inanother step the microvesicles are enriched and differentiated to soproduce a primary result based on one or more distinct RNA molecules, asecondary result based on at least two distinct RNA molecules, and/or aternary result based on a sub-segregated proxysome population and atleast one of the plurality of distinct RNA molecules. In yet anotherstep, the primary, secondary, and/or ternary results are correlated witha stage of the neoplasm in the mammal.

Generally all neoplasms are deemed suitable for use herein (supra),however, it is particularly preferred that the neoplasm is characterizedby overexpression (relative to healthy cell) of ERRB2. Consequently,suitable neoplasms include acute lymphoblastic leukemia, bladder cancer,brain cancer, breast carcinoma, cervical cancer, colorectal cancer, lungcancer, ovarian cancer, and pancreatic adenocarcinoma. RNA coding forERRB2 overexpression can be quantified in numerous manners, however, itis generally preferred that the step of differentiating comprisesquantitative rtPCR and/or reverse transcription and solid phase (e.g.,microarray) hybridization. As already noted above, it is generallypreferred that the step of correlating comprises comparison of theprimary, secondary, and/or ternary results with one or more referenceresults that are characteristic of a healthy cell and/or a diseasedcell, tissue or organ in a specific diseased, compromised, or agedstate.

Viewed from a different perspective, contemplated uses of diagnostictests and compositions include diagnosis, predisposition, and prognosisof the metabolic state of a cell, tissue, and/or organ, analysis of theresponse of a cell, tissue, and/or organ to a physical and moretypically chemical stimulus, and/or diagnosis or determination ofpredisposition for a disease or disorder in patient harboring a cell,tissue, and/or organ from which the proxysomes were analyzed. Inespecially preferred uses, the methods according to the inventivesubject matter are employed in the diagnosis of cancer, a pre-cancerouscondition or predisposition, and/or a clinical stage of a cancer.Additionally, it should be noted that contemplated methods are alsodeemed suitable for prenatal diagnosis as fetal microvesicles have beenidentified in (Kidney International, 2007; 72(9):1095-102) amnioticfluid, placenta, and possibly also blood of the pregnant mother.

In still further contemplated uses, it should be appreciated thatmicrovesicle/proxysome compositions and methods according to theinventive subject matter can be used in bioinformatic analysis in which‘normal’ proxysome quantity and composition is acquired and thencompared to proxysome quantity and composition from cells treated withone or more agents or conditions. Wile contemplated compositions andmethods typically reply on already known specific RNAs and additionalmarkers and information bearing components, it should be appreciatedthat new specific RNAs and new information bearing component can berelatively easy obtained. For example, a cell culture of diseased cellsor cells from a disease model can be propagated in vitro. Thesupernatant is then collected and microvesicles are isolated from thesupernatant. In an optional step, protein is extracted from themicrovesicles and antibodies generated. The antibody collection is thensubtracted against normal tissue to obtain disease specific antibodies.Alternatively corresponding experiments can be performed in silico basedon expression profiling, or subtractive nucleic acid libraries ofdiseased and corresponding healthy cells can be generated.

In still further contemplated aspects, the microvesicle diagnosticmethods presented herein can be advantageously employed in drugdiscovery where multiple cell cultures are separately exposed tomultiple drugs. Genomics and proteomics analyses can be performed asknown in the art. However, in preferred aspects, proxysomes can becollected from the individual cultures and analyzed as described above.The so obtained results can then be followed in vivo to ascertain thatin vitro findings correlate with prior in vitro results. Moreover, asthe microvesicles can be collected multiple times from a single animaland as a single collection is sufficient to analyze distinct reactionsin multiple organs without sacrificing speed or accuracy (or theanimal), drug discovery using proxysomes dramatically increasesthroughput in drug discovery. While it is generally preferred thatanalysis is performed using specific RNA(a) and additional informationbearing components, it is also contemplated that themicrovesicle/proxysome components can be analyzed in relative andabsolute proportions to obtain a measure for a particular clinicalparameter. It should still further be noted that while contemplatedmethods and compositions are particularly useful for clinical diagnosticpurposes and R&D, the methods and compositions according to theinventive subject matter will may also be advantageously employed inpersonalized medicine and personalized nutrition in which the effect ofadministration of nutraceuticals (and medications) on microvesicles andproxysomes can be monitored in a highly simplified manner.

It should be appreciated that contemplated tests allow forstandardization, which is one of the more significant hurdles inclinical diagnosis. In one aspect of the inventive subject matter, theRNA signal is normalized against a second signal, typically derived fromthe additional information bearing components (e.g., total signal fromproxysome membrane [e.g., via labeled annexin V]). Alternatively, arange of normal can be established from a clinically healthy population,again using a signal from an additional information bearing component.For example, one could employ a known microvesicle ELISA (which measuresamount of PS, phosphatidylserine), which is highly correlated withmicrovesicles. There is already normal range established in healthyindividuals (about 10 Nm/ml), which may serve as a normalization signal.In such assay, RNA Her2 could be calculated per total microvesicles,measured PS, or HER2 RNA signal per (phosphatidylserine - Plateletmicrovesicles phosphatidylserine). If using realtime PCR, one wouldcompare the number of cycles per phosphatidylserine. In case off anarras, the array signal is correlated to per phosphatidylserine.

Examples

The following is provided to illustrate exemplary methods, conditions,and embodiments in connection with contemplated methods andcompositions, but should not be deemed limiting the inventive subjectmatter.

Detection of Her-2 Expression in Breast Cancer Cell Lines

Real time PCR: Three cancer cell lines were cultured in normal cellculture conditions (37° C., 5% CO2) according to respective ASTMstandard protocols. Total RNA was isolated from the cells using thecommercially available RNeasy isolation kit (QUIAGEN) according tomanufacturer's protocol. After isolation, the concentration of total RNAwas determined by UV spectroscopy using well established methods. Theconcentration of RNA was adjusted for all groups to 50 ng/mcl. Soprepared RNA was reverse transcribed to cDNA using a commerciallyavailable reverse transcription kit (Applied Biosystems) following themanufacturer's protocol. This cDNA was then used for real time PCRstudies. The real time PCR reaction contained Sybr green master mix,forward primer, reverse primer, water, and respective cDNA samples, andthe following HER-2 primers were used (5′- to 3′-end):

Forward: ATTTCTGCCGGAGAGCTTTGAT (SEQ ID NO: 1) Reverse:CCGGCCATGCTGAGATGTATAG (SEQ ID NO: 2)

Real time PCR reaction was performed with using Real Time PCR System ABI7500 (Applied Biosystems), using following settings: 50° C. 2 min, 95°C. 5 min, 95° C. 15 sec, 60° C., 60 sec; Last two cycles were repeated40 times. Relative quantitation of Her-2 mRNA expression was calculatedwith the comparative Ct method. The relative quantitation value oftarget, normalized to an endogenous control B2MG gene and relative to acalibrator, is expressed as 2^(−ΔΔCt) (fold difference), where ΔCt=Ct oftarget gene, −Ct of endogenous control gene (B2MG), and ΔΔCt=ΔCt ofsamples for target gene-ΔCt of calibrator for the target gene.

To avoid the possibility of amplifying contaminating DNA (i) all theprimers for real time PCR were designed with an intron sequence insidecDNA to be amplified, (ii) all reactions were performed with appropriatenegative controls (template-free controls), (iii) a uniformamplification of the products was rechecked by analyzing the meltingcurves of the amplified products (dissociation graphs), and (iv) gelelectrophoresis was performed to confirm the correct size of theamplification and the absence of unspecific bands. The results for thisexperiment are shown in FIG. 1. As can be readily taken from the Figure,and as assessed via rtPCR, SKOV3 exhibited significant expression ofHer-2 whereas Her-2 expression in T47D cells was moderate and nearlyundetectable in MCF cells. This finding correlates well with publishedliterature data on Her-2 expression in these cell lines.

Regular PCR: Using RNA isolated as described above, a regular PCR wasperformed. Briefly, RNA was reverse transcribed to cDNA with usingApplied Biosystem reverse transcription kit following the manufacturer'sprotocol, and the so prepared cDNA was used for PCR studies. Regular PCRreaction used the following Her-2 primers (5′- to 3′-end):

Forward: GTGACAGCAGAGGATGGAACAC (SEQ ID NO: 3) Reverse:CGCCATTGTGCAGAATTCG (SEQ ID NO: 4)

To avoid the possibility of amplifying contaminating DNA (i) all theprimers for PCR were designed with an intron sequence inside cDNA to beamplified, and the (ii) reactions were performed with appropriatenegative controls (template-free controls). The PCR products werevisualized using a 1.5% agarose gel. Once more, Her-2 was detectable inthe mRNA level in all three cell lines, but SKOV3 cell lines showedhighest expression, and T47D cells and MCF cells showed expression onvery low level. Negative controls were performed using RNA withoutreverse transcription, and PCR products were not detected.

Detection of Her-2 in Microvesicles from Supernatants of the Cell Lines

Isolation of Microvesicles: The tumor derived microvesicles wereisolated from culture conditioned media. Briefly, the supernatant wasspun at about 850 g for 10 minutes at 4° C. The supernatant wascollected and the pellets were discarded. The supernatants were againspun at 24,000 g for 2 hr at 4° C. and the supernatant was discarded.PBS supplemented with HEPES (5 mM) was added to pellet that nowcontained microvesicles and the resuspended microvesicles weretransferred to Eppendorf tubes (1.6 ml) in which they were spun atmaximum speed for 60 min at 4° C. The supernatant was discarded and PBSsupplemented with HEPES was added to the pellet, spun again at maximumspeed for 60 min at 4° C. The so obtained pellet was resuspended pelletin a small volume of PBS supplemented with HEPES (usually 100-200 mcl).

PCR Analysis: Using the methods described for cells above, total RNA wasisolated from the microvesicles, and real time PCR and regular PCR wereperformed. When using real time PCR, Her-2 was detected only inmicrovesicles from the SKOV cell culture but not from T47D and MCF cellculture. However, when using regular PCR, Her-2 was detectable on themRNA level from all three cell cultures by agarose gel electrophoresisas can be seen in FIG. 2A. Not surprisingly, a quantitative analysis ofthe agarose gel electrophoresis showed that microvesicles from the SKOVcell culture provided the strongest her-2 signal, which was followed bythe T47D cell culture, which was in turn followed by the MCF cellculture. A quantitative graph illustrating these results is shown inFIG. 2B. It should therefore be appreciated that cellular expressionlevels of Her-2 are paralleled by microvesicular Her-2 RNA quantitiesdetected in the culture supernatants of the respective cell lines.

Detection of Her-2 in Microvesicles from Murine Serum of Mice HarboringSKOV Derived Tumors

Mice with SKOV-derived Tumors: Athymic nude mide were injected with3×10⁶ SKOV3 cells using standard protocol to establish solid tumors inthese mice. After three weeks, presence of palpable/measurable tumorswas confirmed, and serum was collected from the animals as well as fromcontrol mice without tumors (no SKOV injection).

The microvesicles were isolated from the serum following the generalprotocol as outlined above. The total RNA obtained from themicrovesicles was evaluated for the presence of Her-2 RNA using the PCRprotocols as provided above. PCR products were quantified and checkedvia agarose gel electrophoresis and exemplary results are shown in FIG.3A (to ensure analysis of equal amounts, control PCR was performed usingbeta-2-microglobulin as standard). A graphic representation of the Her-2quantification of corresponding rtPCR is shown in FIG. 3B. Based onthese results, it should be appreciated that SKOV-tumor derivedmicrovesicles that were generated in vivo were not only positive forHer-2 RNA, but that such RNA can also be detected from a relativelysmall blood sample with a high degree of sensitivity and specificity.

Detection of Her-2 in Microvesicles from Human Serum of PatientsDiagnosed with Her-2 Positive Breast Cancer

To validate the concept of using microvesicles as proxy diagnosticmarkers for diseased or otherwise distressed cells, blood samples wereanalyzed from patients diagnosed with Stage I and II breast cancer thatwas characterized by immunohistochemical assay as Her-2 positive atlevel 3+.

Microvesicles were isolated from actual patient and control serafollowing the general protocol as outlined above, and total RNA obtainedfrom the microvesicles was evaluated for the presence of Her-2 RNA usingthe PCR protocols as provided above. The PCR products were quantifiedand checked via agarose gel electrophoresis and the results for theseexperiments are shown in FIG. 4A (to ensure analysis of equal amountscontrol PCR was again performed using beta-2-microglobulin as standard).Remarkably, none of the control sera from healthy volunteers showed anydetectable Her-2 RNA as tested by rtPCR and normal PCR, but all three ofthe patients with confirmed breast cancer exhibited a significant andstrong signal. Moreover, while patients in lane 4 and 6 were staged atStage I, the patent in lane 5 was diagnosed at Stage II, whichcorrelated with the strongest signal. The corresponding graphicrepresentation of the Her-2 quantification is shown in FIG. 4B. Based onthese results, it should be appreciated that microvesicles can act assensitive proxy diagnostic tools for the cells from which theyoriginate. Still further, it is noted that such analysis can beperformed not only the without radiation burden from radiographicanalyses, but also in a manner that entirely avoids any biopsies

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc. Furthermore, where a definitionor use of a term in a reference, which is incorporated by referenceherein is inconsistent or contrary to the definition of that termprovided herein, the definition of that term provided herein applies andthe definition of that term in the reference does not apply.

1. A method of analyzing a biological sample of a mammal, comprising:obtaining a sample comprising a plurality of microvesicles that includea plurality of distinct RNA molecules from a living donor mammal;enriching and differentiating the microvesicles to produce at least oneof (1) a primary result based on at least one of the plurality ofdistinct RNA molecules, (2) a secondary result based on at least two ofthe plurality of distinct RNA molecules, and (3) a ternary result basedon a sub-segregated proxysome population and at least one of theplurality of distinct RNA molecules; and correlating at least one of theprimary, secondary, and ternary results with a diagnosis or prognosis ofa condition of the mammal.
 2. The method of claim 1 wherein the at leastone of the plurality of distinct RNA molecules in the primary result isan RNA that is overexpressed or mutated, and wherein the overexpressionor mutation is characteristic for the condition.
 3. The method of claim1 wherein one of the at least two of the plurality of distinct RNAmolecules in the secondary result is an RNA that is overexpressed ormutated, and wherein the overexpression or mutation is characteristicfor the condition, and wherein the other of the at least two of theplurality of distinct RNA molecules in the secondary result is an RNAthat is uniquely expressed in a specific tissue or organ of the mammal.4. The method of claim 1 wherein the sub-segregated proxysome populationis obtained by isolating the proxysome population based on a surfacemolecule specific for the origin of the proxysome population, whereinthe at least one of the plurality of distinct RNA molecules in theternary result is an RNA that is overexpressed or mutated, and whereinthe overexpression or mutation is characteristic for the condition. 5.The method of any one of claim 2, claim 3, or claim 4, wherein the RNAthat is overexpressed or mutated encodes (1) at least one of MMP11(Matrix metalloproteinase 11), BCAR1 (Breast cancer anti-estrogenresistance 1 protein), ERBB2 (v-erb-b2 avian erythroblastic leukemiaviral oncogene homolog 2; Her2/neu), MKI67 (Antigen identified bymonoclonal antibody Ki-67), PLAU (Plasminogen activator), and TP53(Tumor protein p53) where the condition is breast cancer; (2) at leastone of FGFR1 (Fibroblast growth factor receptor 1), KRAS2 (v-Ki-ras2Kirsten rat sarcoma 2 viral oncogene homolog), TGFBR2 (Transforminggrowth factor, beta receptor II), MAP2K4 (Mitogen-activated proteinkinase kinase 4), and CDKN2A (Cyclin-dependent kinase inhibitor 2A)where the condition is pancreatic cancer, (3) at least one of KLK3(Kallikrein 3), ERBB2 (v-erb-b2 avian erythroblastic leukemia viraloncogene homolog 2), FGF8 (Fibroblast growth factor 8), PSCA (Prostatestem cell antigen), and CAV1 (Caveolin-1) where the condition isprostate cancer, (4) at least one of BAX (BCL2-associated X protein),SLC2A1 (Solute carrier family 2, member 1), PTGS2 (Cyclooxygenase-2),MUC1 (mucin 1), and RUNX3 (Runt-related transcription factor 3) wherethe condition is gastric cancer, (5) at least one of BCL10 (B-cellCLL/lymphoma 10), PAP (Pancreatitis-associated protein), SPARC(osteonectin), CD44 (CD44 antigen), and TP53 (Tumor protein p53) wherethe condition is liver cancer, or (6) at least one of an adult stem cellmarker selected from the group consisting of CD33, CD44, CXCR4, CXCR4+,lin-, CD45−, Oct-4, Nanog, SCA1, 7-AAD where the condition is a cancer.6. The method of claim 3 wherein the RNA that is uniquely expressed inthe specific tissue or organ encodes (1) at least one of DCD(dermcidin), SCGB2A2 (secretoglobin, family 2A, member 2), and ANKRD30A(ankyrin repeat domain 30A) where the specific tissue or organ is breasttissue, (2) at least one of UCN3 (urocortin 3), IPF1 (insulin promoterfactor 1, homeodomain transcription factor), and REG1B (regeneratingislet-derived 1 beta) where the specific tissue or organ is a pancreas,(3) at least one of UPK3A (uroplakin 3A), SEMG1 (semenogelin I), andPRAC (small nuclear protein PRAC) where the specific tissue or organ isprostate tissue, (4) at least one of GAST (gastrin), GKN1 (gastrokine1), and TFF2 (trefoil factor 2) where the specific tissue or organ isgastric tissue, or (5) at least one of GYS2 (glycogen synthase 2), F9(coagulation factor IX), and HRG (histidine-rich glycoprotein) where thespecific tissue or organ is a liver.
 7. The method of claim 1 whereinthe step of enriching the microvesicles is performed using a step ofaggregation in which the microvesicles are interlinked with aninterlinking composition.
 8. The method of claim 7 wherein theinterlinking composition comprises annexin V, fibrin, or an antibody orfragment thereof against at least one of a tetraspanin, ICAM-1, andCD86.
 9. The method of claim 1 wherein the primary or secondary resultis obtained using a quantitative rtPCR for the at least one of theplurality of distinct RNA molecules.
 10. The method of claim 1 whereinthe living donor mammal is a human, and wherein the step of obtainingcomprises collecting plasma or serum.
 11. The method of claim 10 whereinthe diagnosis is a cancer, a pre-cancerous condition, or a clinicalstage of a cancer.
 12. The method of claim 1 wherein the microvesicleshave a membrane composition such that phosphatidylserine is on theoutside of the membrane.
 13. A method of staging a mammalian neoplasm,comprising: obtaining a whole blood fraction that includes a pluralityof microvesicles comprising a plurality of distinct RNA molecules;enriching and differentiating the microvesicles to produce at least oneof (1) a primary result based on at least one of the plurality ofdistinct RNA molecules, (2) a secondary result based on at least two ofthe plurality of distinct RNA molecules, and (3) a ternary result basedon a sub-segregated proxysome population and at least one of theplurality of distinct RNA molecules; and correlating at least one of theprimary, secondary, and ternary results with a stage of the neoplasm inthe mammal.
 14. The method of claim 13 wherein the mammalian neoplasm isselected from the group consisting of acute lymphoblastic leukemia,bladder cancer, brain cancer, breast carcinoma, cervical cancer,colorectal cancer, lung cancer, ovarian cancer, and pancreaticadenocarcinoma.
 15. The method of claim 13 wherein one of the at leasttwo of the plurality of distinct RNA molecules in the secondary resultor the at least one of the plurality of distinct RNA molecules in theprimary or ternary result is an RNA that encodes ERBB2.
 16. The methodof claim 13 wherein the step of enriching is performed by centrifugationor by aggregation in which the microvesicles are interlinked with aninterlinking composition.
 17. The method of claim 13 wherein the step ofdifferentiating comprises quantitative rtPCR.
 18. The method of claim 13wherein the step of correlating comprises comparing the at least one ofthe primary, secondary, and ternary results with a reference result.